Part:BBa_K3559014
BioBrick_BASIC_gRNA_Aro7_1
A gRNA targeting the yeast ARO7 promoter.
Biology
Aro7 is the Saccharomyces cerevisiae gene encoding the enzyme chorismate mutase (E.C.5.4.99.5). Chorismate mutase is the enzyme responsible for the conversion of chorismate to prephenate, which is the first step in the phenylalanine/tyrosine specific branch of aromatic amino acid synthesis. This branch of aromatic amino acid synthesis therefore competes with biosynthesis of the other aromatic amino acid L-Tryptophan.
This particular gRNA targets 15bp from the transcription start site of Aro7, as it is intended to target close to the Aro7 promoter for maximal transcriptional knockdown.
An additionally interesting piece of biology is the ribozymes contained within this sequence. These are RNA motifs that form secondary structures with catalytic activity. The specific ribozymes in this part the Hammerhead and HDV ribozymes, can carry out cleavage reactions at a specific location within their own RNA molecule. This makes these sequences useful for insulating parts from their transcriptional context to ensure they function as intended.
Usage
This part was aimed to be used in Imperial College's 2020 iGEM Tryptophan optimisation project. We reasoned that knocking down the competing aromatic amino acid pathway would lead to increased yields of L-tryptophan. We believe this part would be of interest to any teams looking to also optimise tryptophan biosynthesis using a dCas9 knockdown approach.
Characterisation
As a result of restricted lab access due to the Covid pandemic, we were unable to test this part in the lab. We therefore can make no comment on whether this part functions as intended.
Sequence and Features
While we expect part will function as intended using BioBrick assembly as a single part, there are a number of additional features within the sequence. Firstly, the part is immediately flanked by the BASIC integrated prefix (iP) and the BASIC integrated suffix (iS), thus allowing teams to make use of BASIC assembly as opposed to BioBrick assembly should they wish. Secondly, the sgRNA itself is surrounded by a hammerhead ribozyme and an HDV ribozyme. This means that post transcriptionally the sgRNA will be cleaved from the remainder of the transcript. This means multiple of these type parts could be assembled together to produce gRNA arrays, where several gRNAs are transcribed and then released from the original transcript via the ribozyme activity. We therefore believe these parts are powerful tools for allowing easy multiplexed transcriptional regulation.
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 163
Illegal NgoMIV site found at 192 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 8
Illegal BsaI.rc site found at 236
| None |